When a plasmid containing a target sequence of interest is transformed into a plant, testing needs to take place to confirm that the transformation has occurred and assess the quality of the transformation. For example, when selecting among multiple plants having been transformed with the same construct, the plant selected should have the intact target sequences of interest without rearrangements, insertions, deletions, or extraneous flanking sequences.
Historically, southern blot methods have been used to confirm the transformation of plasmid constructs and identify potential rearrangement, multi-copy, or partial events. Southern blot experiments can be time-consuming, provide low-resolution, have high cost, and require multiple manual inspections. In addition, southern blot methods are not able to identify the target sequence integration site, nor the flanking sequences of the integration site which are useful for designing an event-specific PCR experiment.
Flanking sequence analysis (FSA) has been successfully used to identify the transgene integration site and to obtain the flanking sequences of the insertion site. Nevertheless, as FSA only targets limited border regions, FSA does not detect potential partial fragments, rearrangements, or truncations beyond the targeted border regions. In addition, the search algorithm utilized by FSA can identify false positives caused by any errors in the reference sequence of the plasmid.
Thus, a continuing need exists for a rapid, low-cost method to effectively characterize the location, number, and integrity of target sequence insertions into plant genomes.